Using the polymerase chain reaction to maintain DNA probe inventories in clinical and diagnostic laboratories

Clinical and diagnostic DNA laboratories must maintain a large inventory of DNA probes for use in hybridization studies. The preparation of plasmid DNA and isolation of DNA fragments for use as probes in both expensive and time consuming. We present here a rapid and relatively inexpensive method of producing large amounts of DNA fragments from stocks, using the polymerase chain reaction (PCR). Our experience over the past year using this technique has been very positive and we believe many laboratories could benefit by employing such a labor-saving approach to maintaining DNA probes. The technique uses the bacteriophage M13 DNA sequencing primers to amplify cloned inserts contained in commonly used plasmid vectors. As examples, we illustrate the use of DNA produced in this manner as probes for linkage analysis of the fragile X syndrome and for detection of deletions in the Duchenne muscular dystrophy gene. We have also found that at least two probes can be amplified in the same PCR reaction, allowing the detection of two different restriction fragment length polymorphisms (RFLP) simultaneously. It should be possible for laboratories to devise strategies particular to their individual needs using more than one DNA probe produced in the same PCR reaction to detect RFLP's. Such strategies would need only to consider that the predicted alleles of the multiple polymorphisms do not migrate to the same position during electrophoresis. Stocks of single or multiple probes produced by the PCR could then be maintained for more rapid Southern analyses.     

Hemophilia A: genetic prediction and linkage studies in all available families in Finland

RFLP studies were done in 82 (75%) of all known hemophilia A families in the Finnish population (approximately 5 million). Two intragenic RFLPs (Bc1I/F8A, XbaI/p482.6) and two extragenic markers (TaqI/St14, Bg1II/DX13) were used. Among 263 females at risk, carriership could be evaluated with an intragenic marker in 47% and with an extragenic marker in 26%. In 27% of the females, carriership could be neither excluded nor confirmed; 68% of these females were relatives of an isolated patient. Eight recombinations between the factor VIII gene (F8C) and DXS52 (lod 25.02 at theta max 0.06), eight recombinations between F8C and DXS15 (lod 21.91 at theta max 0.05), and two recombinations between DXS52 and DXS15 (lod 33.56 at theta max 0.01) were found. Using multipoint linkage analysis, the most likely order of loci supported by the data was: F8C-DXS15-DXS52-DXS134. RFLP segregation analysis provides a highly useful method of carrier detection and prenatal diagnosis of hemophilia A, but its limitations must be carefully taken into account.     

解放军医学图书馆电子阅览室的技术特点

从阅览室网络拓扑结构、硬盘保护、网络控制、网址限制及视频点播等几个方面对电子阅览室采用技术的特点逐一做了简要介绍,并以此阐明通过综合管理,使阅览室各计算机能够正常、安全的运行,从而达到既可为读者提供优质服务又可保证系统安全的目的.     

Architectural alterations in rat cerebral microvessels after hypobaric hypoxia

We performed 3-dimensional studies of vascular casts of the microvasculature of the cerebral cortex of rats that were exposed to three weeks of hypobaric hypoxia and of control rats. Scanning electron microscopy of the casts gave the qualitative impression of increased vascularity of the cerebral cortex, particularly the deeper layers, in hypoxic rats. Quantitative analysis of capillary segment lengths revealed a significant shift in the frequency distribution to longer lengths (from 77 ± 8 to 90 ± 14 μm) in the deep, but not in the superficial, layers of the cerebral cortex of hypoxic rats. These findings agree with previous results reporting increased capillary density in the brain after exposure to prolonged hypobaric hypoxia and suggest that capillary segment elongation plays a role in the increased capillary density in the deeper layers of the cerebral cortex.     

269例10万元以上住院费用及影响因素分析

目的分析高额住院费用的成因及影响因素,以控制医疗费用的过快增长.方法选择某综合医院2002年住院费用在10万元以上的病例共269例,分析其费用构成,并对其影响因素进行多元回归分析.结果269例病人的平均费用为159 765元,以治疗费最高,所占比例为51.32%,药费其次,所占比例为36.25%.住院费用的影响因素为:住院天数、院内感染和预后.结论降低过高的住院费用要缩短无效住院日、控制院内感染以及减少无效的治疗和用药.     

巩膜环扎加压术后眼轴与屈光状态的改变

目的：探讨视网膜脱离巩膜环扎加压术后眼部屈光状态的改变。方法：用眼科A超、检影验光等对86例原发性视网膜脱离患者的86只患眼手术前1d及术后1、4、12w时的眼轴长度、眼屈光度等进行测量并将结果进行比较。结果：眼轴长度在术后1、4、12w时均较术前增长(P＜0．01)。眼屈光度在术后1、4、12w时均较术前增加，且向负值偏移(术后1、4w，P＜0．015术后12w，P＜0．05)。结论：巩膜环扎加压术后眼轴长度和屈光状态的变化与眼内嵴有关，术后眼轴增长是眼屈光度负值增加的主要因素。     

High level production of soluble single chain antibodies in small-scale Escherichia coli cultures

We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15–25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80–150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20°C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.     

Human placental growth hormone

Placental growth hormone is the product of the GH-V gene specifically expressed in the syncytiotrophoblast layer of the human placenta. Placental growth hormone differs from pituitary growth hormone by 13 amino acids. It has high somatogenic and low lactogenic activities. Assays by specific monoclonal antibodies reveal that in the maternal circulation from 15 to 20 weeks up to term placental growth hormone gradually replaces pituitary growth hormone, which becomes undetectable. It is secreted by the placenta in a nonpulsatile manner. This continuous secretion appears to have important implications for physiologic adjustment to gestation and especially in the control of maternal insulin-like growth factor-I levels. Placental growth hormone secretion is inhibited by glucose in vitro and in vivo and is significantly decreased in the maternal circulation in pregnancies with intrauterine growth restriction. Placental growth hormone does not appear to have a direct effect on fetal growth because this hormone is not detectable in the fetal circulation. However, the physiologic role might also include a direct influence on placental development through an autocrine or paracrine mechanism, as suggested by the presence of specific growth hormone receptors in this tissue.(Am J Obstet Gynecol 1997;177:1526-34.)     

红色素基因RFLP及其与情感性精神病关系的研究

以红色素基因全长ｃＤＮＡ作探针，２０例正常人，２６例双相型情感性精神病患者进行ＲＦＬＰ分析。ＳａｃＩ酶切显示：部分正常人和部分患者基因组ＤＮＡ产生的７．５ｋｂ，４．８ｋｂ，４．５ｋｂ呈多态性改变的片段。正常人具多态改变的占４５％，患者为４６．２％，按Ｘ染色体数计，正常人出现７．５ｋｂ限制性片段的频率为１４．３％，患者为３２．４％，但两者差异不显著。     

HLA-DR, DQ genotypes of celiac disease patients and healthy subjects from the West of Ireland

Celiac disease (CD) has one of the strongest class II HLA associations of any human illness. We used DNA-RFLP typing to study the class II HLA genotypes of celiac disease patients from the West of Ireland, the geographic area with the highest rate of celiac disease in the world. We confirmed the high frequency of HLA-DR3 in this population, and we were also able to demonstrate the additional risk of developing celiac disease imparted by HLA-DR7. This was done by clearly distinguishing DR7, DQ2 haplotypes from DR7, DQ9 haplotypes, and by "subtraction analysis" of haplotype frequencies. As reported in other populations, most of the patients without DR3 were heterozygous for DR7 and DR11 or 12 (DR5), or had DR4. We used PCR-RFLP and direct sequencing of amplified DNA to examine HLA-DR4 subtypes. The frequency of HLA-DR4 was markedly decreased in patients compared with controls (p=0.000001) and there was a significant alteration of DR4 subtypes of the patients compared with controls (p=0.0227). Moreover, all of the CD patients (5 of 5) with DR4 had a haplotype associated with the DQB1*0302 allele compared with only 11 of 23 control subjects with DR4. Our results in this population with exceptionally high risk of CD strongly support the DQ heterodimer hypothesis and suggest that the recently described sequence difference between the DQB1*02 alleles of DR3 and DR7 may contribute to a synergistic increased risk when these haplotypes are inherited together. In addition, our findings suggest a role for HLA-DQ in DR4-associated CD.